Comparison of Effects of p53 Null and Gain-of-Function Mutations on Salivary Tumors in MMTV-Hras Transgenic Mice

RESEARCH ARTICLE

Comparison of Effects of p53 Null and Gain-of-Function Mutations on Salivary Tumors inMMTV-Hras Transgenic MiceDadi Jiang1¤, Catherine I. Dumur2,3, H. Davis Massey2,3, Viswanathan Ramakrishnan4,Mark A. Subler1, Jolene J. Windle1,3*

1 Department of Human and Molecular Genetics, Virginia Commonwealth University, Richmond, Virginia,United States of America, 2 Department of Pathology, Virginia Commonwealth University, Richmond,Virginia, United States of America, 3 Massey Cancer Center, Virginia Commonwealth University, Richmond,Virginia, United States of America, 4 Department of Public Health Sciences, Medical University of SouthCarolina, Charleston, South Carolina, United States of America

¤ Current address: Department of Radiation Oncology, Stanford University, Stanford, California, UnitedStates of America* [email protected]

Abstractp53 is an important tumor suppressor gene which is mutated in ~50% of all human cancers.

Some of these mutants appear to have acquired novel functions beyond merely losing wild-

type functions. To investigate these gain-of-function effects in vivo, we generated mice of

three different genotypes: MMTV-Hras/p53+/+, MMTV-Hras/p53-/-, and MMTV-Hras/p53R172H/R172H. Salivary tumors from these mice were characterized with regard to age of

tumor onset, tumor growth rates, cell cycle distribution, apoptotic levels, tumor histopatholo-

gy, as well as response to doxorubicin treatment. Microarray analysis was also performed

to profile gene expression. The MMTV-Hras/p53-/- and MMTV-Hras/p53R172H/R172H mice

displayed similar properties with regard to age of tumor onset, tumor growth rates, tumor

histopathology, and response to doxorubicin, while both groups were clearly distinct from

the MMTV-Hras/p53+/+ mice by these measurements. In addition, the gene expression pro-

files of the MMTV-Hras/p53-/- and MMTV-Hras/p53R172H/R172H tumors were tightly clus-

tered, and clearly distinct from the profiles of the MMTV-Hras/p53+/+ tumors. Only a small

group of genes showing differential expression between the MMTV-Hras/p53-/- and MMTV-

Hras/p53R172H/R172H tumors, that did not appear to be regulated by wild-type p53, were iden-

tified. Taken together, these results indicate that in this MMTV-Hras-driven salivary tumor

model, the major effect of the p53 R172H mutant is due to the loss of wild-type p53 function,

with little or no gain-of-function effect on tumorigenesis, which may be explained by the tis-

sue- and tumor type-specific properties of this gain-of-function mutant of p53.

PLOS ONE | DOI:10.1371/journal.pone.0118029 February 19, 2015 1 / 23

OPEN ACCESS

Citation: Jiang D, Dumur CI, Massey HD,Ramakrishnan V, Subler MA, Windle JJ (2015)Comparison of Effects of p53 Null and Gain-of-Function Mutations on Salivary Tumors in MMTV-Hras Transgenic Mice. PLoS ONE 10(2): e0118029.doi:10.1371/journal.pone.0118029

Academic Editor: Hirofumi Arakawa, NationalCancer Center Research Institute, JAPAN

Received: September 9, 2014

Accepted: January 4, 2015

Published: February 19, 2015

Copyright: © 2015 Jiang et al. This is an openaccess article distributed under the terms of theCreative Commons Attribution License, which permitsunrestricted use, distribution, and reproduction in anymedium, provided the original author and source arecredited.

Data Availability Statement: Microarray data usedin this study are deposited in the Gene ExpressionOminibus (GEO) database with accession #GSE59452. (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE59452)

Funding: Support was provided by the Departmentof Defense Breast Cancer Research Program - grant# BC981128 (to JJW) [http://cdmrp.army.mil/funding/bcrp.shtml]; VCU Massey Cancer Center Pilot ProjectGrant - no # (to JJW and MAS) [https://www.massey.vcu.edu/research/funding/mcc-pilot-project-program/];and Start-up funds (to JJW). The funders had no role

Introductionp53 is widely regarded as one of the most important tumor suppressor genes, as it is mutatedin over 50% of all human cancers, and the p53 pathway is found to be inactivated in most if notall tumors [1]. p53 knock-out mice are largely normal at birth, indicating a non-essential rolefor p53 during embryonic development. However, the mice are highly tumor-prone, with themajority developing lymphomas and sarcomas by 6 months of age, underscoring p53’s essen-tial tumor suppressor function [2–5]. The p53 protein executes its tumor-suppressive functionsprimarily through its role as a sequence-specific transcription factor [6]. Upon stabilizationand activation by oncogenic signals or other types of cellular stresses, including DNA damage,hypoxia, nutrient deprivation and reactive oxygen species (ROS), p53 transactivates or transre-presses a panel of downstream effector genes which are involved in mediating multiple cellularresponses, including transient G1 cell cycle arrest, DNA repair, cellular senescence character-ized by a permanent cell cycle arrest, and apoptosis [2,7].

Most p53 mutations arise somatically during tumor development and progression. Howev-er, p53 mutations can also be transmitted in the germ-line and give rise to cancer predisposi-tion conditions called Li-Fraumeni Syndrome (LFS) and Li-Fraumeni-like Syndrome (LFL)[8–10]. Unlike changes in other classical tumor suppressor genes during tumorigenesis, whichfrequently involve frame-shift or nonsense mutations, nearly three-quarters of both somaticand germ-line p53 mutations are missense mutations, according to the latest release (R17) ofthe IARC p53 mutation database [1] (also see http://www-p53.iarc.fr/). The vast majority ofthese missense mutations are clustered in the central DNA-binding domain of the p53 protein,either affecting residues directly contacting DNA (DNA contact mutations), or those impor-tant for maintaining the conformation of the DNA-binding domain (conformational or struc-tural mutations). Both classes of mutations disrupt the sequence-specific DNA-bindingfunction of the p53 protein, thus preventing p53 from acting as a transcription factor [11,12].In addition to loss of the wild-type functions, some p53 mutants also demonstrate dominant-negative effects over the remaining wild-type allele, preventing the wild-type p53 protein frominhibiting cellular transformation [13,14]. Further, a subset of p53 missense mutants have beenshown to possess a variety of novel gain-of-function properties. When expressed in a p53-nullbackground, these p53 mutants confer accelerated cell growth in vitro [15–21], increasedtumorigenicity in mouse xenograft models [16,20,22–24], anti-apoptotic effects and chemore-sistance [25–30], exacerbated genomic instability [25,31–33], enhanced somatic cell program-ming [34], disruption of tissue architecture [35], and increased migration, invasion andmetastasis [16,36,37]. Although the mechanism(s) contributing to these gain-of-function ef-fects are still under investigation, several models have been proposed. Firstly, a subset oftumor-derived p53 mutants physically interact with a host of cellular proteins such as p63/73,MRE11, PML and Pin1 [38]. Interaction between mutant p53 and the p53 family membersp63/73 leads to altered activities of these sequence-specific transcription factors and contrib-utes to promotion of chemoresistance, migration, invasion and metastasis [36,37]. Alternative-ly, mutant p53 may also transcriptionally regulate a novel set of genes, many of which areinvolved in increasing cell proliferation, inhibiting apoptosis, promoting chemoresistance, andregulating metabolism as well as cell-cell/cell-ECM signaling pathways [13,38–40]. The alteredtarget affinity in transcriptional regulation by mutant p53 is postulated to be mediated throughinteraction with other sequence-specific transcription factors, thus inducing or repressing theirtarget gene expression.

Although the majority of studies characterizing the gain-of-function properties of mutantp53 have been conducted using cell culture systems, a variety of genetically engineered mousetumor models have also been developed for examining the effects of mutant p53 in vivo. For

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in study design, data collection and analysis, decisionto publish, or preparation of the manuscript.

Competing Interests: The authors have declaredthat no competing interests exist.

example, when p53 R172H, which is equivalent to the hot-spot human p53 R175H mutant (aconformational mutation), was expressed in the epidermis [41] or mammary glands [42] oftransgenic mice, accelerated carcinogen-induced skin or mammary tumorigenesis was ob-served in the corresponding mouse models. These mutant p53-expressing tumors also demon-strated features of advanced malignancy when compared to wild-type p53-expressingcounterparts. Knock-in mouse models of p53 R172H and R270H (a hot-spot DNA contact mu-tant, equivalent to human R273H) have also been generated by two different groups, allowingfor a comparison of the effects of null versus gain-of-function mutants on the tumor phenotypeof these mice [45,46]. Although survival time was not significantly different between mice har-boring the mutant p53 alleles and p53-deficient mice, mutant p53 knock-in mice displayed analtered tumor spectrum, with a greater number of mice developing carcinomas, and the tumorsthat arose metastasized with a much higher frequency [43,44].

Although the mutant p53 knock-in mice allowed for the comparison of tumorigenesis andtumor properties in mice carrying p53 null and p53 gain-of-function alleles, direct compari-sons within a particular tumor type were complicated by the fact that mice in these models de-velop multiple tumor types. We therefore sought to create animal models that would allow fora head-to-head comparison of the effects of p53 null and gain-of-function mutations withinthe context of a single tumor type. Our lab previously utilized the MMTV-v-Ha-ras (MMTV-Hras) transgenic mouse mammary/salivary tumor model [45] crossed with p53 null mice [3]to study the influence of p53 loss on tumorigenesis and tumor properties. While the MMTV-Hras/p53+/+ mice develop both mammary and salivary tumors, salivary tumorigenesis wasgreatly accelerated in the MMTV-Hras/p53-/- mice. The MMTV-Hras/p53-/- salivary tumorshad higher histopathological grades, growth rates, S-phase fractions, and genomic instabilitythan the MMTV-Hras/p53+/+ tumors [46]. In addition, the MMTV-Hras/p53+/+ tumors re-sponded better to doxorubicin treatment than the MMTV-Hras/p53-/- tumors, due to a de-crease in S phase fraction and an increase in G1 phase fraction, effects which were absent in thetreated MMTV-Hras/p53-/- tumors [47]. No significant difference in apoptotic levels was iden-tified in this tumor model, regardless of the p53 status and treatment, likely due to the anti-apo-ptotic effects of the activated Ras signaling pathways driving tumorigenesis in this model.

In the current study, we have included p53 R172H as the third p53 status in this tumormodel by crossing theMMTV-Hrasmice to p53 R172H knock-in mice [46], and have per-formed head-to-head comparisons of the effects of the three classes of p53 status on salivary tu-morigenesis, tumor properties, and tumor response to doxorubicin. While the MMTV-Hras/p53+/+ tumors differed significantly from both the MMTV-Hras/p53-/- and MMTV-Hras/p53R172H/R172H tumors in these measurements, p53 null and R172H tumors exhibited very sim-ilar properties. We also performed gene expression profiling on salivary tumors of the three ge-notypes and identified the differentially regulated genes among the three classes. Again, thep53+/+ tumors clustered separately from the other two classes, but there were relatively fewgene expression differences between the p53 null and R172H tumors. These results indicatethat, within the context of activated Ha-ras expression in the mouse salivary gland, the primaryeffect of p53 R172H mutation is the loss of wild-type p53 function, with little discernable gain-of-function effect on tumorigenesis.

Materials and Methods

Ethics StatementAll animal studies and care were performed under the guidelines of the Virginia Common-wealth University (VCU) Institutional Animal Care and Use Committee (IACUC), in accor-dance with the principles and procedures outlined in the National Research Council “Guide for

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the Care and Use of Laboratory Animals” under Assurance Number A3281-01. Specific ap-proval for these studies was granted by the VCU IACUC under protocol #AM10313, entitled“Molecular Genetics of Tumorigenesis in Transgenic Mice”. Pain and distress resulting fromtumor development was minimized by euthanizing any mouse with a palpable tumor of2500mm3 or displaying indications of morbidity.

Generation of MMTV-Hras, MMTV-Hras/p53-/- and MMTV-Hras/p53R172H/R172HmiceMMTV-Hras and MMTV-Hras/p53-/- mice were generated as described in [46], and have beenmaintained on a Balb/c x C57BL/6 (CB6) genetic background for>30 generations (includingmultiple generations of breeding of colony mice to purchased CB6F1 mice.) p53R172H/+ micewere generously provided by Dr. Gigi Lozano (The University of Texas MD Anderson CancerCenter) [44]. To generate MMTV-Hras/p53R172H/R172H mice, p53R172H/+mice on a C57BL/6background were initially mated to MMTV-Hras/p53+/- mice on a CB6 background. The colo-ny was subsequently maintained on the C57BL/6 x BALB/c mixed background by interbreed-ing of mice from this colony. Mice were genotyped by PCR for determination of ras transgeneand p53 status [44,46].

Identification of salivary tumor onset and measurement of tumor weightMice were monitored visually or by palpation 2–3 times weekly for the presence of tumors aris-ing from the salivary glands (parotid, submandibular, and sublingual glands). Once a tumorwas detected, tumor size was measured (3 times per week initially and daily as the tumor be-came larger) by caliper measurement of the width and length of the tumor. Tumor weight wasestimated using the formula: tumor weight (mg) = (L x W2)/2, where L is the length andW isthe width of the tumor in mm. Tumor-bearing mice were sacrificed when the weight of thetumor approached 2500 mg (or smaller for some studies) and the tumor was dissected out. Thetumor mass was then cut into multiple sections, one of which was placed in 10% formalin andthe rest into 1.2 ml cryovials which were then flash frozen in liquid nitrogen and stored at -80°.

Analysis of age of tumor onset and tumor growth ratesAge of tumor onset data were recorded for all male MMTV-Hras/p53+/+, MMTV-Hras/p53-/-,and MMTV-Hras/p53R172H/R172H mice. The survival function was used to calculate the salivarytumor-free survival for each group of mice using GraphPad Prism 4. Mice that died or weresacrificed without developing a salivary tumor were censored in the survival analysis. Only tu-mors with initial palpable size<500 mg were used to calculate mean tumor weights for plottingtumor growth curves.

Immunoblotting and immunohistochemistryFor immunoblotting, tumor protein was extracted from frozen tissue using PIERCE RIPAbuffer, supplemented with additional 1X protease inhibitor cocktail containing 50mMNaF,1mMNa3VO4, 17.5mM β-glycerophosphate, and 1mM PMSF (Calbiochem). For immunohis-tochemistry, tumor tissues fixed in 10% phosphate-buffered formalin were transferred to70% ethanol after 48 hours to preserve antigens. Fixed tumor tissues were paraffin embeddedand cut into 5μm sections for staining. Primary antibodies and dilution used for immunoblot-ting include: p53 (CM5, Novocastra) at 1:1000; p19ARF (ab80, Abcam) at 1:1000; p16 (M-156, Santa Cruz Biotech) at 1:1000; p15 (K-18, Santa Cruz Biotech) at 1:1000 and Actin(C-11, Santa Cruz Biotech) at 1:1000. HRP-conjugated Secondary antibodies (Jackson

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ImmunoResearch Laboratories) were used at 1:2000 dilution. An enhanced chemilumines-cence (ECL) assay (PerkinElmer) was used and documented either on Kodak X-Omat BlueXB-1 film or by an AlphaEase digital imaging system (Alpha Innotech). Primary antibodiesand dilution used for immunohistochemistry include: Ki-67 (Clone SP6, Lab Vision) at 1:200;cleaved caspase 3 (Biocare Medical) at 1:100; p53 (CM5, Novocastra) at 1:100.

Characterization of tumor response to doxorubicinA subset of salivary tumor-bearing mice in each genotypic group were treated with doxorubi-cin. When the weight of the tumor reached 500–700 mg, the mouse was subjected to a 9-daytreatment schedule at a dose of 2 mg/kg doxorubicin/day, injected IP. During the treatment pe-riod, tumor dimensions were measured daily and tumor weight calculated the same way as inthe tumor growth analysis. Only tumors with initial palpable size<800 mg were used to calcu-late mean tumor weights for plotting tumor growth curves.

Gene expression profiling by microarray analysisFor RNA extraction, 20–40 10 μm frozen sections from each candidate tumor were collected.RNA was extracted using TRIZOL reagent (Invitrogen Life Technologies), according to themanufacture’s protocols. Total RNA isolated from the tumor sections was subjected to a clean-up step using the RNeasy Mini Kit (Qiagen). Microarray analysis was performed using theAffymetrix GeneChip platform and the Mouse 430A 2.0 array according to the Affymetrixstandard protocol. Multiple levels of data analysis were performed in the BRB-ArrayTools soft-ware, version 3.5.0 (Richard Simon & Amy Peng Lam, Biometric Research Branch, Division ofCancer Treatment and Diagnosis, NCI). The probe-level microarray data (in .CEL files) werecollated in BRB-ArrayTools using the RMA (Robust Multi-chip Average) method. Microarraydata used in this study are deposited in the Gene Expression Ominibus (GEO) database withaccession # GSE59452.

Validation of microarray data by quantitative PCR3 μg of total RNA from each tumor sample was loaded in each RT reaction, using the Super-Script III First-strand Synthesis system (Invitrogen Inc.) according to the manufacture’s proto-col, and oligo(dT) was selected to prime the RT reactions. Primers for the real-time PCR assayswere designed using the Primer Express Version 3.0 software (Applied Biosystems) using thestandard melting temperature (60°C) and at least one of the two primers for each gene was se-lected to span an exon-intron boundary, to avoid amplifying genomic DNA contaminants. Thereal-time PCR assay was performed on the Applied Biosystems 7500 Real-time PCR System(Applied Biosystems). The Relative Standard Curves method was used as the study design forthe quantitative PCR assay (Real-Time PCR Systems Chemistry Guide, Applied Biosystems).β-actin was used as the internal control. The raw data were analyzed in the Sequence DetectionSoftware Version 1.3.1 (Applied Biosystems) according to the manufacture’s protocol. Se-quences of the primers used in the quantitative PCR assays are listed in S7 Table.

Results

Age of tumor onset for mice of each p53 genotypeTo determine the potential differential effects of p53 loss-of-function and gain-of-function mu-tations on tumor phenotype within the context of a single tissue of origin (salivary tumors aris-ing in MMTV-Hrasmale transgenic mice), we generated cohorts of mice of the followinggenotypes: MMTV-Hras/p53+/+, MMTV-Hras/p53-/-, and MMTV-Hras/p53R172H/R172H.

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Although female MMTV-Hras/p53+/+ mice develop primarily mammary tumors, male mice ofthis genotype develop primarily salivary tumors (at a later average age of tumor onset). Howev-er, we have previously shown that when bred to p53 knock-out mice, both males and femalesof the MMTV-Hras/p53-/- genotype develop primarily salivary tumors due to an accelerationof salivary but not mammary tumorigenesis in this model [46]. We observed a similar preva-lence of salivary tumorigenesis in the MMTV-Hras/p53R172H/R172H mice. Therefore, in order tocompare tumors of a single cell type of origin arising in mice of a single gender, we restrictedour analysis to salivary tumorigenesis in male mice of each of the three genotypes. (A smallnumber of female mice were also used for some of the analyses, and no recognizable differencein the salivary tumor properties characterized between the two genders was observed.)

S1 Table summarizes the mice used for the age of tumor onset analysis. Significant fractionsof the p53-/- and p53R172H/R172H mice developed symptoms indicating the presence of lympho-mas and/or sarcomas prior to the development of salivary tumors, consistent with previous re-ports of the prevalence of these tumor types in p53-/- and p53R172H/R172H mice lacking theMMTV-Hras transgene [2–5,43,44], which necessitated sacrifice of the mice. Because thetumor spectrum in both p53-/- and p53R172H/R172H mice has already been well-characterized[3,4,43,44,48], we did not analyze tumor types in animals lacking salivary tumors. In addition,38 MMTV-Hras/p53+/+ mice (of a total of 114) without salivary tumors were sacrificed due todevelopment of Harderian gland tumors, consistent with previous reports [45]. Animals devel-oping lymphomas, sarcomas or Harderian gland tumors were censored from the dataset usedfor Kaplan-Meier analysis. With a similar total starting number of mice in each genotypicgroup, the mean ages of salivary tumor onset in the MMTV-Hras/p53-/- and MMTV-Hras/p53R172H/R172H mice were much earlier than in the MMTV-Hras/p53+/+ mice and the overallsalivary tumor incidence was much higher than in the MMTV-Hras/p53+/+ mice. As shown inFig. 1 and S1 Table, the median time to salivary tumor onset was 177 days for the MMTV-Hras/p53+/+ mice, 68 days for the MMTV-Hras/p53-/- mice and 109 days for the MMTV-Hras/p53R172H/R172H mice. We observed a statistically significant difference between the MMTV-Hras/p53-/- and MMTV-Hras/p53R172H/R172H mice in age of tumor onset (p = 0.0261, Log-ranktest), with a slightly delayed tumor onset in the MMTV-Hras/p53R172H/R172H mice. However,both the MMTV-Hras/p53-/- and MMTV-Hras/p53R172H/R172H mice developed salivary tumorsmuch more rapidly than the MMTV-Hras/p53+/+ mice (p<0.0001, Log-rank test).

To exclude the possibility that tumors from the MMTV-Hras/p53+/+ mice had acquired so-matic mutations in the p53 gene which might confound our subsequent analyses, exons 5–9 ofthe p53 locus of 4 MMTV-Hras/p53+/+ salivary tumors (all of which were used for the subse-quent microarray analysis) were sequenced, and no p53mutations were detected (data notshown).

Growth rates for tumors of each p53 genotypeWe have previously shown that salivary tumors arising in MMTV-Hras/p53-/- mice havegrowth rates that are nearly double those of salivary tumors arising in MMTV-Hras/p53+/+

mice [46]. To determine whether a p53 gain-of-function mutation further accelerates thetumor growth rate, the growth of multiple independent salivary tumors arising in mice of eachof the three different genotypes (MMTV-Hras/p53+/+, MMTV-Hras/p53-/-, and MMTV-Hras/p53R172H/R172H) was compared (Fig. 2; S1 Fig.). Tumor growth data from 26 MMTV-Hras/p53+/+ tumors (16 from males and 10 from females), 34 MMTV-Hras/p53-/- tumors (21 frommales and 13 from females), and 39 MMTV-Hras/p53R172H/R172H tumors (35 from males and4 from females) was obtained. The mean tumor growth curves for each genotype are shown inFig. 2, and the individual tumor growth curves are shown in S1 Fig. The growth of most of the

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salivary tumors roughly followed an exponential growth pattern, which indicates the existenceof constant doubling times of these tumors. The MMTV-Hras/p53+/+ tumors grew significantlyslower than the MMTV-Hras/p53-/- and MMTV-Hras/p53R172H/R172H tumors, as was previous-ly reported for the comparison of MMTV-Hras/p53+/+ andHras/p53-/- tumors [46]. However,there was no significant difference between the growth rates of theHras/p53-/- and MMTV-Hras/p53R172H/R172H tumors.

p53 protein levels in tumors of each genotypeTo confirm that mutant p53 was expressed and stabilized in the MMTV-Hras/p53R172H/R172H

tumors, and to characterize the expression patterns of mutant p53 protein in the tumor tissues,western blotting and immunohistochemical analysis were performed on tumors of each geno-type, using an antibody that detects both wild-type and mutant p53. Western blot analysisshowed that mutant p53 was present in very high levels in all of the MMTV-Hras/p53R172H/R172H

tumors tested, whereas p53 was barely detectable in the MMTV-Hras/p53+/+ tumors andcompletely absent in the MMTV-Hras/p53-/- tumors (Fig. 3A). Immunohistochemical stainingwith the same antibody on the tumor sections showed that while p53 was undetectable in bothMMTV-Hras/p53+/+ and MMTV-Hras/p53-/- tumors, the MMTV-Hras/p53R172H/R172H tumorsdisplayed a relatively uniform, high level of p53 in the nucleus of tumor cells, but not in the adja-cent stromal cells (Fig. 3B).

Fig 1. Kaplan-Meier analysis of salivary tumor-free survival. The proportion of tumor-free mice as a function of time for MMTV-Hras/p53+/+ (green),MMTV-Hras/p53-/- (yellow), and MMTV-Hras/p53R172H/R172H (red) male mice are shown, based upon the age at which a palpable salivary tumor wasfirst detected.

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Tumor histologyWe have previously shown that in this MMTV-Hras transgenic mouse tumor model, absenceof p53 resulted in tumors with higher histologic grades [46]. To determine whether p53 loss-of-function and gain-of-function mutations differentially affected tumor histopathology, for-malin-fixed tumor samples from the three genotypic groups were embedded in paraffin blocks,and sections were stained with hematoxylin and eosin and subjected to microscopic evaluation.A set of morphological parameters were evaluated using a 3-point grading system. We assessedthe nuclear/cytoplasmic ratio, the degree of nuclear pleomorphism, and overall tumor architec-ture. We also took in account the presence of a spindle cell morphology, and whether “giantcells”, which display remarkably enlarged cell and nucleus areas, were present. Mitotic activity,percentage of tumors with apoptotic cells, and degree of necrosis were also estimated. The re-sults are summarized in S2 Fig. In general, tumors of each of the three genotypes were well-lo-calized and encapsulated with no signs of metastasis, and were composed of nests and cords ofpoorly differentiated carcinoma cells. The MMTV-Hras/p53+/+ tumor cells were relatively uni-form in size and shape, while both the MMTV-Hras/p53-/- and MMTV-Hras/p53R172H/R172H

tumor cells displayed more variability in cell and nuclear size and shape and less evenly distrib-uted chromatin in the nucleus (Fig. 4). When nuclear/cytoplasmic ratio, level of pleomorphism,

Fig 2. Tumor growth rates. Tumor growth rates over time (days) measured as calculated weight (mg) foreach of the three groups of tumors were plotted. Each data point represents the mean ± SEM.

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and cellular architecture were measured using a 3-point grading system, the MMTV-Hras/p53+/+ tumors were assigned a lower grade on average than the MMTV-Hras/p53-/- andMMTV-Hras/p53R172H/R172H tumors, although the differences were not statistically significant(S2 Fig. A). The MMTV-Hras/p53+/+ tumors contained fewer cells with mitotic figures thanthe MMTV-Hras/p53-/- and MMTV-Hras/p53R172H/R172H tumors, while the latter two groupswere indistinguishable (S2 Fig. E). It is also noticeable that there were high levels of variation inall of the four measurements, indicating the existence of significant heterogeneity in the histo-pathology of these tumors. Proportions of tumors displaying features of spindling (S2 Fig. B)and apoptosis (S2 Fig. D), as well as those containing “giant cells” with greatly enlarged cell andnuclear size are also summarized (S2 Fig. C). Similarly higher fractions of the MMTV-Hras/p53-/- and MMTV-Hras/p53R172H/R172H tumors demonstrated features of spindling than theMMTV-Hras/p53+/+ tumors. Interestingly, the “giant cells” represented a slightly higher frac-tion of the tumor cells in the MMTV-Hras/p53R172H/R172H tumors than in the other two groupsof tumors. There was no significant difference in the fractions of tumors demonstrating apoptosisamong the three genotypic groups. As the levels of apoptosis in individual tumors were not takeninto account in this analysis, the result has limited value as to measure the representative apopto-tic levels. The levels of necrosis were also estimated. Compared to the MMTV-Hras/p53+/+ andMMTV-Hras/p53-/- tumors, the MMTV-Hras/p53R172H/R172H tumors displayed higher levels ofnecrosis (S2 Fig. F).

Cell cycle and apoptosis studiesWe previously showed that MMTV-Hras/p53-/- tumors had a significantly higher fraction ofcells in the S and G2/M phases of the cell cycle, with a corresponding decrease in the fraction ofcells in G1, compared to MMTV-Hras/p53+/+ tumors [46]. To compare the effect of p53 loss-of-function and gain-of-function mutations on tumor cell proliferation, tumors of each geno-type were immunohistochemically stained with an antibody for the proliferation marker Ki-67.

Fig 3. p53 protein levels in tumors of each genotype. (A) Western blot analysis of p53 using an antibody that detects both wild-type and mutant p53(CM5). From left to right: 3 MMTV-Hras/p53+/+, 3 MMTV-Hras/p53-/-, and 4 MMTV-Hras/p53R172H/R172H tumors. (B) Immunohistochemical staining with theCM5 anti-p53 antibody in a representative tumor of each of the three genotypes.

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Fig 4. H&E staining of representative tumor of the three genotypes. Top: MMTV-Hras/p53+/+; middle:MMTV-Hras/p53-/-; bottom: MMTV-Hras/p53R172H/R172H.

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However, there was extensive heterogeneity in the Ki-67 staining across the entire set of tumorsamples. Frequently, tumors from the same genotypic group (S3 Fig. A), or even different re-gions of the same tumor (S3 Fig. B), displayed highly variable fractions of Ki-67-positive cells.The staining ranged from a complete lack of staining to staining of almost every cell. This vari-ation hampered accurate assessments of the representative fraction of proliferating cells of eachgenotypic group. However, there were no grossly recognizable differences in the overall stain-ing patterns of the three groups of tumors.

Previous studies also demonstrated that apoptosis levels were constitutively low in the tu-mors with the MMTV-Hras transgene regardless of whether they were p53+/+ or p53-/-, likelydue to the anti-apoptotic effects of the pathways downstream of activated Ras [46]. To investi-gate whether the same scenario holds true for tumors bearing the p53R172H mutation, we exam-ined the levels of spontaneous apoptosis in the three groups of tumors by staining the tumorsimmunohistochemically with an antibody specific for cleaved caspase 3 (Cc3), which is the ac-tivated version of a key apoptosis mediator. As for the Ki-67 staining, there was also significantvariation in the staining patterns for Cc3, either among different tumors, or among differentregions of the same tumor. Cc3 immunostaining revealed low indices of apoptosis in all threegenotypic groups of tumors (data not shown).

Tumor response to doxorubicinIt is well established that p53 mutation confers resistance of tumor cells to a wide range of che-motherapeutic agents [38], and several studies have demonstrated increased chemoresistancein tumors expressing gain-of-function p53 mutants compared to those lacking p53 altogether[17,20,21,25,29]. We have previously shown that tumors arising in MMTV-Hras/p53-/- miceare significantly impaired in their response to doxorubicin compared to those arising inMMTV-Hras/p53+/+ mice [47]. To determine whether the p53 R172H gain-of-function muta-tion promoted further resistance to doxorubicin, a subset of salivary tumor-bearing mice fromeach of the three genotypic groups was treated with doxorubicin for 9 consecutive days, andthe effects on tumor growth were measured (Fig. 5; S4 Fig.). As we previously reported, thegrowth of MMTV-Hras/p53+/+ tumors plateaued following the first day of treatment, withsome of the tumors actually regressing during the 9-day treatment period. In contrast, both theMMTV-Hras/p53-/- and MMTV-Hras/p53R172H/R172H tumors continued to grow with unal-tered rates for 3–4 days, after which the growth of most of the tumors began to plateau. Al-though the average size of the MMTV-Hras/p53R172H/R172H tumors at the initiation oftreatment was larger than that of the MMTV-Hras/p53-/- tumors, the overall shape of thetumor response curves was nearly superimposable. Interestingly, 3 of the 18 MMTV-Hras/p53R172H/R172H tumors in the treatment study exhibited no plateau phase and continued togrow at an unaltered rate during the 9-day treatment period (S4 Fig.).

Gene expression profiling of tumors with different p53 statusTo compare the gene expression profiles of tumors with different p53 genotypic status andidentify genes differentially regulated, 4 tumors from each genotypic group were analyzedusing Affymetrix GeneChip oligonucleotide arrays. After the model-based expression summa-ries were obtained, an unsupervised hierarchical clustering analysis of the 12 tumor sampleswas conducted and is summarized in Fig. 6A. As expected, the 4 samples with wild-type p53fell into a tight cluster, which indicates close similarity within the gene expression profiles ofthis group. On the other hand, this unsupervised clustering method failed to separate theMMTV-Hras/p53-/- or MMTV-Hras/p53R172H/R172H tumors into distinct groups, signifying thelack of significant numbers of genes that are consistently differentially expressed between the

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p53-null and mutant p53 tumors. This suggests that the primary effect of the R172H mutationis the disruption the DNA-binding domain and subsequent gene-regulation capacity of wild-type p53.

As our major interest was to identify differentially expressed genes that may indicate gain-of-function effects, we compared the expression profiles of the three groups of tumors, and tu-mors with wild-type p53 were used to control for residual wild-type functions of mutant p53.To identify the genes differentially expressed between the three groups, we performed classcomparison analysis using Significance Analysis of Microarray (SAM). The multi-classcomparison function of SAM gave rise to the identification of 188 genes with a low 1% FalseDiscovery Rate (FDR). The 188 genes were then subjected to hierarchical clustering to identifyco-expressed gene clusters, the result of which is shown in Fig. 6B. By visually examining thepattern of the expression levels across the three genotypic groups, the 188 genes were furthercategorized to 6 groups (i to vi). The expression levels and potential meaning of the 6 clustersof genes are summarized in S2 Table and the 188 genes are listed in S3 Table. Of the 188 genes,184 show a clear difference in expression levels between samples with wild-type p53 and with-out p53 (except the 4 genes in Cluster iii), which represent the differentially expressed genesbetween the wild-type p53 (WT) and p53-null status in this analysis. Of these 184 genes, only a

Fig 5. Tumor growth responses to doxorubicin. Tumor-bearing mice were treated for nine consecutive days with doxorubicin (2 mg/kg), and tumor growthwas monitored daily by caliper measurements. Tumor growth rates over time (days) measured as calculated weight (mg) for each of the three groups oftumors were plotted. Each data point represents the mean ± SEM.

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few were previously reported as regulated by wild-type p53, including 3 well-characterized p53target genes Cdkn1a (p21),Mdm2, and Ccng1. These genes are listed in S4 Table. To explorepotential biological pathways in which these differentially expressed genes may function, this184 gene-signature was submitted to Ingenuity Pathway Analysis, which identifies the mostsignificantly perturbed signaling and metabolic pathways, molecular networks, and biologicalprocesses. The most highly enriched biological functions from the analysis are summarized inS5 Table. The top two networks identified are displayed in S5 Fig. The first network involves bi-ological functions including Cancer, Cell Cycle, and Developmental Disorder, with a score of37 defined by IPA and 20 focus molecules, where p53 sits right at the center. The second net-work is enriched for biological functions including Cell Death, Dermatological Diseases andConditions, and Cancer, with a score of 34 and 19 focus molecules.

Of the 6 gene clusters, Cluster iii and v define the genes with higher expression levels in themutant p53-expressing samples than in the wild-type p53-expressing and p53-null samples (S2Table), which could potentially represent genes contributing to the gain-of-function propertiesof the p53 R172H mutant. As many of these genes have anti-apoptotic and cell migration-pro-moting activities (see DISCUSSION), these two clusters represent good candidates for thegain-of-function properties of the p53 R172H mutant, including insensitivity to treatment-in-duced cell death and elevated metastatic potential. Mutant p53-expressing samples retained ex-pression levels similar to those samples with wild-type p53 (Cluster iv), or intermediate levels(Cluster i and vi) for a significant fraction of the remaining genes. This indicates the presenceof residual wild-type functions with the mutant p53 in regulating gene expression. However,when examining the overall expression profiles based on these 188 genes, the two groups show-ing the greatest similarity were the mutant p53-expressing tumors and p53-null tumors, whileboth showed significant differences from the tumors with wild-type p53, in agreement with theresults from the unsupervised clustering of the samples (Fig. 6A). To further support this no-tion, we performed class comparison analysis between MT-p53 and p53-null samples, as wellas between wild-type p53 and p53-null samples, using three widely-used probe-level data-pro-cessing algorithms (RMA, MBEI, and MAS5) and two False Discovery Rate (FDR)-based statis-tical approaches (SAM and student t-test with Benjamini-Hochberg correction). From thisanalysis we found that despite the different scales of significant genes identified by differentmathematical and statistical approaches, there are consistently very few genes standing outfrom the comparisons between MT-p53 and p53-null samples, in sharp contrast to thosebetween wild-type p53 and p53-null samples (S6 Table). This demonstrates that in thisMMTV-Hrasmouse salivary tumor model, the p53R172H mutant status shares a very similargene expression profile with p53-null status.

To validate the microarray experiment, we performed real-time PCR to quantitate the ex-pression levels of Cap1, which is significantly up-regulated in MT-p53 expressing samplescompared to the other two groups of tumors. We also checked the protein levels of p16Ink4aand p19Arf, the two genes expressed from the Cdkn2a locus, which is down-regulated in wild-type p53-expressing samples based on the array analysis. Both the real-time PCR and Western

Fig 6. Gene expression profiling analysis of salivary tumors with different p53 status. (A)Unsupervised hierarchical clustering of MMTV-Hras/p53+/+ (green), MMTV-Hras/p53-/- (yellow), andMMTV-Hras/p53R172H/R172H (red) tumors based on gene expression profiling. (B) Heatmap of the identified188 genes through the multi-class comparison function of SAM with a low 1% False Discovery Rate (FDR)and hierarchical clustering. Red represents higher expression levels and blue represents lower levels,while white represents intermediate levels. The 6 sub-clusters identified through visual inspection aremarked by color bars on the right (i-vi).

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Blotting analyses showed similar results as measured by microarray, which confirmed the va-lidity of the microarray analysis (Fig. 7).

DiscussionBeyond loss of p53 wild-type functions, a subset of p53 mutants gain novel functions promot-ing more severe malignant properties. Although extensive studies on the gain-of-function ef-fects of mutant p53 have been carried out using in vitro cell culture systems, fewer in vivomodels have been developed to investigate these effects under physiological conditions and in anormal cellular context with endogenous cell-cell and cell-ECM interactions. Our study con-tributes to research on the gain-of-function effects of mutant p53 by introducing a mutant p53allele with well-characterized gain-of-function properties into the MMTV-Hras transgenicmouse model, in which deficiency for p53 has been shown to accelerate salivary tumorigenesisand alter tumor properties.

Interestingly, the data from this study demonstrated that MMTV-Hras/p53R172H/R172H andMMTV-Hras/p53-/- mice are very similar with regard to age of salivary tumor onset, tumorgrowth rates, tumor histopathological features, and response to a DNA-damaging agent, doxo-rubicin. This similarity is not due to lack of mutant p53 expression in the MMTV-Hras/p53R172H/R172H salivary tumors, because both western blotting and p53 immunohistochemistryanalysis showed that mutant p53 accumulated to uniformly high levels in the tumors (Fig. 3).Compared to other in vivo analyses on the same p53 mutant through knock-in approach, ourfinding is not surprising. First, in a K-ras-driven lung adenocarcinoma model, neither thep53R172H nor the p53R270H mutant displayed any detectable gain-of-function activity comparedto total p53 loss [49]. In another study,WAP-Cre-induced expression of the p53R270H mutantin p53-null mouse mammary glands caused no difference in tumor latency compared top53-null glands [50]. These observations suggest that tissue specificity plays a role in the invivo activity of these mutants. In addition, knock-in mouse models homozygous for thep53R172H mutation (or one p53R172H allele and one null allele) displayed no significant differ-ence in survival time of the mice when compared to p53-null mice [43,44,51]. One exceptionwas a model expressing a humanized p53R248Q allele, which significantly reduced lifespan whencompared to p53-null mice [52]. In two of the studies [44,51], there was no difference betweenthe tumor spectra of p53R172H/R172H and p53-/- mice, while in the third study [43], p53R172H/-

mice developed carcinomas and hemangiosarcomas with increased incidence compared top53-/- mice. The discrepancy between these studies may be due to the fact that mice with differ-ent genetic backgrounds were used. The common gain-of-function effect observed in all threestudies was that tumors in the p53R172H/+ mice metastasized with a high incidence, a feature thep53+/- tumors lacked.

In this study, with a set of well-defined histopathologic parameters, the MMTV-Hras/p53-/-

tumors displayed more malignant properties than the MMTV-Hras/p53+/+ tumors, consistentwith our previous findings [46]. The MMTV-Hras/p53R172H/R172H tumors closely resemble theMMTV-Hras/p53-/- tumors with regard to most of these parameters, suggesting that loss of thesequence-specific DNA binding function of wild-type p53 is the primary cause of the differencein tumor histology between tumors with mutant and wild-type p53.

In a previous study from our lab [47], the MMTV-Hras/p53-/- tumors displayed a delayedresponse upon doxorubicin treatment, as compared to the MMTV-Hras/p53+/+ tumors.Apoptotic levels were shown to be minimal in both types of tumors, either with or withouttreatment. Instead, doxorubicin treatment caused significant changes in cell cycle distributionin these tumor cells. In the present study, the MMTV-Hras/p53R172H/R172H tumors demonstrat-ed indistinguishable response patterns compared to the MMTV-Hras/p53-/- tumors.

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Immunohistochemical analysis of cleaved caspase 3 (Cc3) revealed similarly low levels of apo-ptosis in tumors of all three genotypes (data not shown). Thus, the results demonstrated againthat apoptosis does not play a major role in tumor properties or response to chemotherapeutic

Fig 7. Validation of the microarray data for sample genes by qPCR and western blotting. (A) Top:Expression levels of theCap1 gene in 4 salivary tumors per group measured by qPCR normalized to β-actin ±coefficient of variance (top), and by microarray analysis (bottom). (B) Protein levels of p16Ink4a, p19ARF, andp15Ink4b in 5 MMTV-Hras/p53+/+ and 5 MMTV-Hras/p53-/- tumors. β-actin was used as a loading control.

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treatments in this model, likely due to the anti-apoptotic effects of the activated Ras signalingdriving tumorigenesis in this model. Thus, it is important to note that, with the low level ofboth spontaneous and drug-inducible apoptosis in this tumor model, it may be difficult to de-tect any influence of mutant p53 on apoptosis, which is an important aspect of the gain-of-function activities identified in in vitro cell culture systems.

One possible explanation for the failure to detect a p53 gain-of-function phenotype distinctfrom the knock-out phenotype could potentially be a lack of stabilization and accumulation ofthe mutant p53 protein. The Lozano group demonstrated that in the R172H knock-in mice,mutant p53 was not stabilized in either normal or tumor tissues, due to the basal level ofMDM2’s inhibitory activity on mutant p53 [53]. They also demonstrated that various types ofstresses that stabilize wild-type p53 also stabilize p53R172H [54]. However, the apparent absenceof a gain-of-function phenotype in our study is not likely due to the stability issue, because mu-tant p53R172H was found to be stabilized in all the tumors tested by both Western blotting andimmunohistochemistry. It is reasonable to speculate that activated Ras signaling is providingthe stabilizing signal for mutant p53 in this tumor model.

Given the similarity between the p53 R172H-expressing and p53-null tumors, but the signifi-cant difference between both groups and tumors with wild-type p53 in the in vivo analyses weperformed, it is not surprising that the gene expression profiles of the different groups of tumorsclosely reflect this relationship. We have tested three prevalent probe-level data summarizationalgorithms for Affymetrix microarray data, and two False Discovery Rate-based class comparisonapproaches, and none of the combinations could detect reasonable numbers of significant genesfrom the comparison between the MMTV-Hras/p53-/- andMMTV-Hras/p53R172H/R172H tumors,while substantial numbers of genes were identified between the MMTV-Hras/p53+/+ tumors andeither the MMTV-Hras/p53-/- or the MMTV-Hras/p53R172H/R172H tumors.

Several mechanisms have been proposed for the gain-of-function activities of mutant p53[38]. One of these mechanisms proposes that mutant p53 actively regulates a unique set ofgenes, the activities of which endow the cell with a growth advantage, chemoresistance, alteredmetabolism and other properties [38]. Unlike wild-type p53, which depends on its sequence-specific DNA binding for its transactivation activity, genes regulated by mutant p53 lack a con-sensus DNA binding site in their promoter regions [38]. It has thus been proposed that insteadof binding to a common response sequence, mutant p53 preferentially binds to stereo-specificDNA configurations [55,56]. Alternatively, mutant p53 may bind to target genes indirectly, i.e.by interactions with other transcription factors, e.g. Sp1 [57,58], Ets [59], NF-Y [21], VDR [60]and SREBP [35]. Although these findings support a transcription-dependent mechanism forthe gain-of-function activities of mutant p53, this hasn’t been tested strictly in an in vivo settingor under physiological conditions. Rather, most of the observations have been made by over-expressing mutant p53 in p53-null cells, and in many cases, in the presence of stress signals.Additionally, almost all of the previous studies only utilized a comparison between cells with-out p53 and those expressing mutant p53, a study design lacking the ability to detect any resid-ual wild-type function of the mutants.

The class comparison analysis in this study identified a small subset of genes with the high-est expression levels in the MMTV-Hras/p53R172H/R172H tumors (Cluster iii and v in Fig. 6; S2Table). We also compared our microarray results to those from other gene expression profilinganalyses designed to identify mutant p53-regulated genes, either from cells cultured in vitro[18,61–63] or tumors from in vivomouse models [64]. One of the mutant p53-induced geneswe identified, Bcl2l1 (Bcl2-like 1, also known as Bclx or BCL-X(L) in human) was previouslyidentified by another group as induced by p53R 175H overexpressed in p53-null H1299 non-small cell lung carcinoma cells. BCL2L1 is a member of the BCL2 family of anti-apoptotic pro-teins [65]. In addition, expression of BCL2L1 was dependent on mutant p53 in SKBR3

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mammary adenocarcinoma cells expressing p53R 175H, and in HT29 human colorectal adeno-carcinoma cells expressing p53 R273H, another p53 mutant with gain-of-function activities[63]. Another gene found to be most highly expressed in the MMTV-Hras/p53R172H/R172H tu-mors, Csnk2a1, which encodes casein kinase 2, alpha 1 polypeptide, has also been shown tohave an anti-apoptotic function, but regulation of its expression hasn’t been associated withmutant p53[66–68]).

Because increased metastatic potential has also been commonly observed with gain-of-func-tion mutants of p53 [43,44], and p53 R175H was shown to promote in vitro cell migration as wellas in vivo tumor metastasis [44], we looked for genes that may contribute to these activitiesamong the list of genes most highly expressed in theMMTV-Hras/p53R172H/R172H tumors. Indeed,a few genes have been reported to promote cell motility and/or metastasis, including Etv4/E1A-F[69,70],Mia1 [71], and Cap1 [72]. Another gene with previously identified association with p53R175H, EFEMP2 (EGF-containing fibulin-like extracellular matrix protein 2) was recognized as abinding partner of mutant human p53 proteins, with the highest specificity to structural mutantslike human p53R175H. Even more intriguingly, co-expression of EFEMP2 and p53 R175H had syn-ergistic effects in promoting neoplastic transformation and tumor cell growth [73]. The fact thatwe didn’t observe a significant decrease in apoptotic levels or response to doxorubicin treatment,and we didn't identify any obvious elevated metastatic potential of the p53 R172H-expressing tu-mors, as compared to p53-null tumors, suggests that these gain-of-function properties may becell/tumor type-specific and depend on specific cellular contexts and molecular events.

It should be noted that although mutant p53 R172H loses its sequence-specific DNA bindingactivity, it still maintains intact N-terminal and C-terminal domains. Thus, an additional pro-posed mechanism for the gain-of-function effects of mutant p53 involves the retention of someresidual wild-type function, which when imbalanced and deregulated may contribute to oppositeeffects [40]. In this study, gene expression profiling also identified a group of genes similarly regu-lated in the MMTV-Hras/p53+/+ andMMTV-Hras/p53R172H/R172H tumors as compared to theMMTV-Hras/p53-/- tumors (Cluster iv and vi in Fig. 6; S2 Table). Although overall the expressionprofiles of the MMTV-Hras/p53R172H/R172H andMMTV-Hras/p53-/- tumors are very similar toeach other (S6 Table), the small subset of genes co-regulated in tumors with wild-type and mu-tant p53 might help elucidate pathways shared by these two functionally very different proteins,and provide insights into the mechanistic basis of the gain-of-function effects.

In conclusion, this study has led to the generation of MMTV-Hras/p53R172H/R172H mice anda comparison between these mice and the previously characterized MMTV-Hras/p53+/+ andMMTV-Hras/p53-/- mice, with regard to salivary tumorigenesis, tumor properties, tumor re-sponses to a chemotherapeutic agent, and tumor gene expression profiles. This study enabledus to compare the influences of different p53 status (wild-type, null, and mutant) on in vivotumor development and test the gain-of-function effects of a p53 mutant in an in vivo setting.Overall, the MMTV-Hras/p53R172H/R172H mice closely resembled the MMTV-Hras/p53-/- micewith regard to age of tumor onset, tumor growth rates, tumor histopathological properties,tumor responses to doxorubicin, and gene expression profiles, while both groups were clearlydistinct from the MMTV-Hras/p53+/+ mice. These results indicate that in this MMTV-Hras-driven mouse salivary tumor model, the primary effect of p53 R172H mutation is the loss ofwild-type p53 function, with little or no gain-of-function effect on tumorigenesis, underscoringthe tissue- and tumor type-specific properties of the gain-of-function mutants of p53.

Supporting InformationS1 Fig. Tumor growth rates. (A-C) Once a tumor was detected, growth was monitored bydaily caliper measurements, and calculated tumor weights (mg) of the three groups of tumors

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were plotted over time. Each line represents the growth of an individual tumor.(TIF)

S2 Fig. Tumor histopathology. Tumor histopathology was graded on a 3-point scale takinginto account (A) N/C (nuclear to cytoplasmic) ratio, degree of nuclear pleomorphism, andoverall tumor architecture; percentage of tumor cells showing (B) spindle cell morphology, (C)“giant cells”, (D) apoptosis, (E) mitotic figures, and (F) necrosis.(TIF)

S3 Fig. Ki-67 staining. (A) Heterogeneity of Ki-67 staining in different tumors from the samegenotypic group. Top: MMTV-Hras/p53+/+; middle: MMTV-Hras/p53-/-; bottom: MMTV-Hras/p53R172H/R172H; left: representative tumors of each genotype with high levels of Ki-67staining; right: representative tumors of each genotype with low levels of Ki-67 staining. (B) Anexample of an MMTV-Hras/p53+/+ tumor in which different regions show markedly differentlevels of Ki-67 staining.(TIF)

S4 Fig. Tumor growth responses to doxorubicin. . (A-C) Calculated weights (mg) of thethree groups of tumors during the treatment period are plotted over time (days). Each line rep-resents the growth of an individual tumor.(TIF)

S5 Fig. The top two networks identified through Ingenuity Pathway Analysis (IPA) on thesignificantly differentially expressed genes. Genes up-regulated in wild-type p53 tumorscompared to p53-null tumors are colored in red and those down-regulated are coloredin green.(TIF)

S1 Table. Summary of the mice used for the age of tumor onset analysis(DOCX)

S2 Table. Overall expression levels and functional meaning of genes in different clusters(DOCX)

S3 Table. List of genes in different clusters(DOCX)

S4 Table. Genes previously reported as regulated by p53(DOCX)

S5 Table. Top enriched biological functions fromWT-p53 vs. p53-null comparison(DOCX)

S6 Table. Number of significant genes from different probe-level data summarization algo-rithms and statistical approaches(DOCX)

S7 Table. Primers used in the quantitative PCR assays(DOCX)

S1 Methods. Supplementary Materials and Methods(DOCX)

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AcknowledgmentsWe thank Dr. Guillermina Lozano for generously providing the p53R172H/+ mice and Dr. Brad-ford Windle for critical reading of the manuscript.

Author ContributionsConceived and designed the experiments: DJ MAS JJW. Performed the experiments: DJ CIDHDM. Analyzed the data: DJ CID HDMVRMAS JJW. Contributed reagents/materials/analy-sis tools: DJ CID HDMVRMAS. Wrote the paper: DJ JJW.

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